The long-term goals of this proposal center on the dissection of the biochemical pathways of sphingolipid metabolism and their role in cell regulation with particular emphasis on "sphingomyelin turnover" and its role in cell differentiation and proliferation. The hypothesis that ceramide functions as a second messenger, lipid mediator of cell differentiation, will be investigated. Control and modulation of cellular responses by extracellular agents involves a number of signal transduction mechanisms. Recent work from this laboratory has resulted in the discovery of a novel mechanism of signal transduction involving the turnover of sphingomyelin, an important cell membrane sphingolipid. This "sphingomyelin cycle" appears to operate in response to certain inducers of cell differentiation (including tumor necrosis factor alpha, gamma-interferon, vitamin D(3) and other steroids and anti-steroids) and it appears to be an important mechanism mediating their effects on cell proliferation/differentiation. The specific aims of this proposal are, therefore, directed at; 1) defining "sphingomyelin turnover" in a number of cell systems and identifying inducers of sphingomyelin hydrolysis; 2) determining the metabolic pathways involved in sphingomyelin turnover with particular attention aimed at purifying and characterizing the regulation of a novel neutral sphingomyelinase; and 3) determining the physiologic role of sphingomyelin turnover and ceramide generation in cell regulation. These studies will establish a role for sphingomyelin hydrolysis in different cellular responses. More importantly, the central hypothesis that the breakdown product of sphingomyelin hydrolysis, i.e., ceramide, functions as a second messenger will be evaluated by; a) measuring ceramide levels in response to inducers of differentiation; b) using cell permeable ceramide analogs as exogenous agents to probe the function of sphingomyelin hydrolysis and ceramide generation; and c) defining the mechanism of action of ceramide with its possible relationship to protein kinase C regulation. These studies are beginning to define a novel area of signal transduction and cell regulation at the biochemical and molecular level with particular attention to its relevance to cell differentiation and regulation of proliferation. This improved understanding may have significant impact in the areas of cancer biology and cell regulation.